A generic standard additions based method to determine endogenous analyte concentrations by immunoassays to overcome...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Immunology

Background:

• Accurate quantification of endogenous analytes in biological matrices is crucial for diagnostics and research.
• Traditional immunoassay methods often require complex standard curves and calibrant diluents, which can be prone to matrix effects.
• Matrix interference in biological samples can significantly impact immunoassay accuracy.

Purpose of the Study:

• To develop a novel, generic method for determining unknown endogenous analyte concentrations in complex biological matrices.
• To overcome limitations of conventional relative quantification methods in immunoassays.
• To account for individual matrix interferences by directly spiking the test sample.

Main Methods:

• A new numerical method is proposed, based on the relationship between immunoassay signal response and log-transformed estimated total analyte concentration.
• The method utilizes a log-log plot where a near-linear relationship indicates correct analyte concentration estimation.
• The technique is analogous to standard additions used for chemical analytes and requires spiking the test sample itself.

Main Results:

• The novel method reliably determines unknown endogenous analyte concentrations in biological samples, including 2-fold diluted human plasma.
• As few as four reaction wells are sufficient for reliable quantification.
• The approach obviates the need for conventional internal standard curves and calibrant diluents.

Conclusions:

• This generic method offers a simplified and accurate approach to analyte quantification in biological matrices.
• The technique effectively addresses matrix interference by incorporating sample-specific spiking.
• It provides a robust alternative for determining endogenous analyte concentrations in various biological samples.